The bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase plays a crucial role in glucose metabolism. It is the sole enzyme responsible for the synthesis and degradation of fructose 2,6-bisphosphate, a potent intracellular modulator of hepatic carbon flux. In liver, the metabolic effects of glucagon, via cAMP-dependent protein kinase, are mediated by the intracellular concentration of fructose-2,6-bisphosphate. High levels of fructose-2,6-bisphosphate allosterically activate the glycolytic enzyme 6-phosphofructo-1-kinase and inhibit the gluconeogenic enzyme fructose-1,6-bisphosphatase, thereby regulating the direction of carbon flux. In experimental (streptozotocin-induced) diabetes in mice, which are devoid of circulating insulin, fructose-2,6-bisphosphate has been shown to appropriately regulate glucokinase (increase) and glucose-6-phosphatase (repress) gene expression in an insulinomimetic manner. In all forms of diabetes, the excessive production of glucose by the liver is a major contributor to hyperglycemia, which leads to the major problems associated with the disease. The central role of fructose-2,6-bisphosphate in control of hepatic glucose production and utilization, as well as gene regulation, suggests that therapies directed toward increasing the fructose-2,6-bisphosphate levels will be beneficial to the diabetic patient. The proposed studies use two approaches to target the bisphosphatase domain of the bifunctional enzyme for inhibition, and thereby, increase the fructose-2,6 bisphosphate levels. First, the metabolic and gene expression effects of manipulation of fructose-2,6-bisphosphate by adenoviral-mediated overexpression of the bifunctional enzyme are being investigated. The overexpression or expression of bifunctional enzyme with deficient bisphosphatase or kinase domains generates high or low levels of hepatic fructose-2,6-bisphosphate, respectively. Using the same arguments outlined above, the inhibition of the kinase activity of the bifunctional enzyme should have opposite effects on hepatic glucose output and should increase blood glucose levels. Second, physical studies using NMR spectroscopy that lead to the characterization of the reaction mechanism and structure of the bisphosphatase domain are underway. These studies will define functional roles of active site amino acid residues during the hydrolysis reaction. Knowledge of this important component of hepatic gluconeogenic/glycolytic flux provides the basis for the rational design of specific inhibitors of the bisphosphatase activity of hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.